摘要
目的制备抗尿激酶型纤溶酶原激活物受体（uPAR）人源化抗体并初步检测它们与抗原的亲和能力。方法通过计算机辅助设计的结果,合成新型抗uPAR抗体的轻链和重链可变区基因序列,通过重叠PCR方法,拼接成完整的轻链和重链基因并克隆入pIRES双向表达载体。瞬时转染293T细胞,收取细胞上清,rProtein A亲和层析法纯化目的抗体,并进行SDS-PAGE和免疫印迹鉴定,采用Biacore3000技术检测抗体与抗原的结合能力。结果成功构建5种表达载体S1~S5,纯化的抗体在还原SDS-PAGE中表现为相对分子质量约为25×103和55×103两条带;免疫印迹分析表明,该人源化抗体可与羊抗人IgG特异性结合。Biacore3000实验结果表明,S2、S4和S5抗体与抗原具有良好的亲和活性,且亲和活性分别为1.74×10-8,1.49×10-8和1.05×10-8mol/L。结论成功构建并表达了5种抗uPAR人源化抗体,其中S2、S4和S5具有良好的抗原结合能力。
Objective To prepare the humanized monoclonal antibodies against urokinase-type plasminogen activator receptor（uPAR）,and preliminarily detect their affinity to uPAR.Methods L and VH genes of humanized monoclonal antibodies against uPAR were designed by the computer and synthesized by overlap PCR.Genes encoding L and H chains were connected and then cloned into vector pIRES,a bicistronic expression vector.The recombinant plasmids were transfected into 293T cells,purified antibodies through rProteinA affinity chromatography,and further confirmed by Western-blotting.The affinity of the humanized antibodies to the uPAR was assessed by Biacore assay.Results Five expression vectors were constructed and the humanized monoclonal antibodies against uPAR were expressed and purified successfully.In reducing SDS-PAGE,the antibodies exhibited two bands of approximately 25×103 and 55×103,respectively.Western blotting assay showed that the humanized antibodies had recognition specificity to goat-human IgG antiserum.Biacore assay revealed that the humanized antibodies S2,S4,and S5 bound to uPAR with high affinity（1.74×10-8mol/L,1.49×10-8mol/L,and 1.05×10-8mol/L,respectively）.Conclusion Five humanized monoclonal antibodies against uPAR have been successfully constructed and expressed,and antibodies S2,S4,and S5 retain high affinity for uPAR.
出处
《军事医学》
CAS
CSCD
北大核心
2011年第4期258-261,共4页
Military Medical Sciences
关键词
受体
尿纤溶酶原激活物
人源化抗体
瞬时表达
receptors
urinary plasminogen activator
humanized antibody
transient expression
